1. Field of The Invention
This invention is in the field of chemotaxis and chronic inflammation.
2. Related Prior Art
The effector cell of chronic inflammation is the macrophage. The macrophage in inflamed tissues is derived from circulating monocytes that originate in the bone marrow. In response to signals from injured tissue, as yet incompletely understood, the monocyte binds to and then migrates beneath the endothelium lining the vascular structures through which it is circulating. Once in a subendothelial position, the monocyte activates and differentiates into a macrophage. The activated macrophage releases a variety of factors that degrade extracellular matrix, stimulate collagen production, and promote proliferation of endothelial cells, fibroblasts, and vascular smooth muscle cells. These factors include proteases, reactive oxygen species, and cytokines such as interleukin-1, tumor necrosis factor, and platelet-derived growth factors, among others, Nathan, C. F. 1987. J. Clin. Invest. 79: 319-326. The end result is the promotion of wound healing or, in the case of inflammation occurring within a bodily organ, cell proliferation and sclerosis that may eventuate in impairment of organ function. Thus the macrophage is central to the pathological processes underlying myocardial fibrosis, atherosclerosis, restenosis, pulmonary fibrosis, progressive nephrosclerosis, arthritis, and inflammatory bowel disease, to cite a few examples of chronic inflammatory conditions, Kissane, J. ed. Anderson's Pathology, 1990. C. V. Mosby, St. Louis, Mo. pp. 89-96, 615-730, 804-871, 920-1047, 1153-1199, 2065-2105.
There are two components of monocyte movement into tissues. The first component consists of monocyte adhesion to the endothelium. There is a growing literature on the expression of adhesion proteins by endothelial and other cells that promote the binding of leukocytes, including monocytes, to the endothelium, Springer, T. 1990. Nature, 346: 425-433. The second component is the signal for chemotaxis, the signal released by traumatized tissue that induces migration of the monocyte beyond the endothelial cell to which it is adhering and into the tissue underlying the endothelium.
The factors regulating the subendothelial migration of monocytes into tissue in normal and inflammatory states are not well understood. Like neutrophils, monocytes display chemotactic migration to C5a, the bacterial tripeptide f-Met-Leu-Phe, proteolytic fragments of collagen and fibronectin, platelet-derived growth factor, transforming growth factor-.beta., and neuropeptides. Schiffman, E., & Gallin, J. 1979. Curr. Top Cell Reg. 15: 203-213; Pierce, G., et al 1989. J. Cell Biol. 109: 429; Snyderman, R., & Mergenhagen S. 1976. In Immunobiology of the Macrophage pp. 323-348, New York Academic Press.
The contribution of the factors cited above to maintaining the mononuclear leukocytic invasion of chronically inflamed tissues has not been defined. Since these factors attract both neutrophils and monocyte/macrophages, and because neutrophils are not a prominent component of chronic inflammatory lesions, there is continuing interest in further identifying chemotactic signals unique to the monocyte or macrophage. The identification of such a chemotactic agent should meet three criteria: 1) it should be chemotactic for monocytes but not neutrophils in vitro; 2) it should be identified in lesions associated with chronic infiltration by monocytes/macrophages; and 3) interruption of its synthesis, release, or receptor activation should be associated with amelioration of chronic inflammation in vivo.
There is only one known chemotactic signal, a protein, unique to the monocyte. It is a lymphocyte-derived, 8 kD, chemotactic protein known as macrophage chemotactic protein, which has been sequenced, Furutani, Y., et al 1989. Biochim. Biophys. Res. Comm. 159: 249. However, no in vivo inhibition of its function has been achieved to date to confirm its role in macrophage migration in vivo.
It has become evident that altered lipid metabolism, particularly hyperlipidemia, may induce or augment monocyte migration into the walls of vascular tissue. The early phase of atherosclerosis is characterized by subendothelial migration of monocytes in the aorta and coronary arteries, Ross, R. 1986. N. Eng. J. Med 296: 488. Hyperlipidemia accelerates the renal infiltration by monocytes in chronic inflammation of the glomerulus and interstitium, Diamond, J., & Karnovsky, M. 1988. Kid. Int. 33: 917. Although there is a clear association between lipidemia and monocyte migration into extravascular spaces, no lipid chemotactic factors have been described that are specific for monocytes at physiological concentrations. LTB.sub.4 is highly chemotactic for polymorphonuclear leukocytes (PMN), much less so for human monocytes, and not all for mouse or rat monocytes, Kreisle, R., et al 1985. J. Immunol. 134: 3356. Platelet activating factor is minimally chemotactic for rat monocytes and more stimulatory for neutrophils, Goetzl, E., et al 1980. Biochim. Biophys. Res. Comm. 94: 881; Rovin, B., et al J. Immunol. A lipid generated during oxidative modification of lipoprotein, lysophosphatidyl choline, possesses modest chemotactic properties for human monocytes, Quinn, M., et al 1988. Proc. Natl. Acad. Sci. 85: 2805, but at extremely high concentrations not found in nature.
To date, there has been no demonstration that in vivo inhibition of chemotactic factors specifically interrupts monocyte migration into tissues. This is true for both the macrophage chemotactic protein as well as for lipid mediators of inflammation.
The model that we have most commonly employed for the study of monocyte chemotaxis in vivo is nephrotoxic serum nephritis, in which rats receive an injection of rabbit anti-glomerular membrane (GBM) antibody. Polymorphonuclear leukocytes (PMNs) enter the glomerulus in the first 3 hours followed by monocytes at 12-24 hours. Schreiner, G., et al 1978. J. Exp. Med. 147: 369. It has been shown that infiltration is not dependent upon complement activation. Schreiner, G., et al 1984, Lab. Invest. 51: 524.
The role of essential fatty acid (EFA) deficiency on the renal infiltration by monocytes of these cells has been studied. Hurd, E., et. al. 1981. J. Clin. Invest. 67: 476, found that NZB/NZW F1 mice with systemic lupus crythematosus did not die of renal failure if placed on a diet deficient in the essential fatty acids, linoleate and arachidonate, despite documented deposits of immune complexes in their glomeruli, and circulating immune complexes. Subsequently it was shown that EFA-deficiency resulted in a marked reduction in the number of resident renal glomerular and interstitial macrophages Lefkowith, J., & Schreiner, G. 1987. J. Clin. Invest. 80: 947. When animals were selectively repleted with (N-6) fatty acid supplementation, it was observed that a spontaneous macrophage repopulation of the glomerulus occurred. It had been previously shown that resident macrophages in the kidney expressing the Ia+ antigene are highly stimulatory in a mixed lymphocyte culture reaction. The effect of whether depletion of these cells from the kidney via this dietary manipulation would decrease the immunogenicity of the kidney when transplanted across a major histocompatibility barrier was studied. Kidneys harvested from a Lewis EFAD donor and transplanted into a Buffalo strain rat on a normal diet survived as allografts in the absence of immunosuppression of the recipient Schreiner, G., et al 1988. Science 240: 1032. Allografts from EFAD donors normalized their lipid composition within the allogeneic recipient and were repopulated with host macrophages within 5 days. The rapid repopulation of the kidney with host macrophages closely paralleled the restoration of the essential fatty acid content of nospholipids, suggesting that the seeding of organs by macrophages could be dependent in part upon a lipid pathway.
The potential role of this lipid pathway in mediating the inflammatory influx of macrophages into the kidney, in acute nephrotoxic nephritis, has been evaluated Schreiner, G., et al 1989. J. Immunol. 143: 3192. The effects of EFA-deficiency were striking. EFA-deficiency completely prevented the influx of macrophages into the glomerulus during the course of the nephritis. In contrast, the preceding PMN influx was unaffected. EFA-deficiency completely prevented polyuria, azotemia, and sodium retention, and largely abrogated the proteinuria. EFA-deficient macrophages are not impaired in their ability to move chemotactically toward either C5a or platelet activating factor; and no circulating inhibitors of chemotaxis were found in EFA-deficient serum, suggesting the effect of EFA-deficiency may be exerted at the level of tissue-derived factors inducing monocyte migration Schreiner, G., et al 1989. J. Immunol. 143: 3192; Rovin, B., et al 1990. J. Immunol 145:1238.
Using our dietary model, Diamond, J., et al 1989. Am. J. Physiol. 257: F798, discovered that animals deficient in essential fatty acids, deficient only during the period of acutely induced nephrotic state by PAN, were protected against the development of glomerular sclerosis four months later. The protection against glomerular sclerosis did not correlate with the degree of proteinuria, hyperlipidemia, or hypertension. Rather, it directly correlated with the inhibition of glomerular macrophage accumulation normally induced by the hyperlipemic state of nephrosis.
It has been observed that EFA-deficiency also prevents the renal mononuclear cell interstitial infiltrate of acute PAN-induced nephrosis and reverses the profound decrease in renal blood flow normally observed in the acute phase of this disease. Parallel experiments with marrow irradiation demonstrated that the predominant effect of EFA-deficiency on preserving renal blood flow and glomerular filtration could be attributed to on blocking mononuclear migration into the interstitium, Harris, K., et al 1990. J. Clin. Invest. 86: 1115.
Importantly, this effect is not confined to the kidney. EFA-deficiency inhibits the development of autoimmune insulitis in mice receiving low dose streptozotocin and in the diabetes-prone BB/Wor rate, Wright, J., et al 1988. Proc. Natl. Acad. Sci. 85: 6137; Lefkowith, J., et al 1989. J. Exp. Med. 161: 729. Both are models of diabetes in which insulitis is preceded by islet infiltration by monocytes. It has been demonstrated that pancreatic islets undergoing oxidative stress after in vivo exposure to streptozotocin release an uncharacterized lipid chemoattractant, specific for monocytes, resembling from that released by isolated glomeruli, Muir, A., et al 1991. Diabetes 40: 1459. In skin graft experiments, we have observed impaired wound healing in fatty acid deficient animals with marked inhibition of monocyte accumulation in the traumatized skin and inhibited formation of granulation tissue (unpublished observations). EFA-deficiency has previously been shown to protect against atherosclerosis, Holman, R. T. 1960. J. Nuir. 70: 405, and inhibit carrageenan-granuloma formation, Bonta, F., et al 1977. Prostaglandins 14: 295, and diminish the leukocyte inflammation associated with experimental myocardial infarction.
The release of a potent uncharacterized chemoattractant for monocytes, Rovin, B., et al 1990. J. Immunol 145: 1238, has been described in short-term cultures of nephritic glomeruli from rats on a standard diet. Production of this chemoattractant is markedly enhanced after induction of nephritis. EFA-deficient nephritic glomeruli do not release the monocyte chemoattractant. In vivo studies employing inhibitors of cyclooxygenase and lipoxygenase have demonstrated that this chemoattractant is not a product of either pathway. Administration of a platelet activating factor (PAF) receptor antagonist similarly failed to inhibit the glomerular influx of macrophage. Lipid (Blight-Dyer) extraction of nephritic glomeruli from control diet animals has yielded chemoattractant activity in the organic phase, Rovin, B., et al 1990. J. Immunol 145: 1238.
These findings suggest that a lipid pathway, metabolically linked to dietary fatty acids, may provide a generalized mechanism for the induction of monocyte infiltration into tissues.
An article by Graziano Guella et al., Helvetica Chimica. Acta. 70; 1050-1059 (1987) describes various long chain acetylenic enol ethers of glycerol derived from marine sponges. The compounds identified, however, are not the same as the lipid chemoattractant of this invention because the compounds described in Guella et al. have properties inconsistent with the properties of the compound of this invention. Specifically, the activity of the Guella et al. compounds, if any, would not be inhibited by selective reducing agents as sodium borohydride. Likewise, the compounds of Guella et al., which are ethers of marine sponges, have never been isolated and purified from urine or plasma of mammals including humans. Furthermore, unlike the lipid chemoattractant of this invention, the Guella et al. compounds would not lose their activity, if they exhibit any, at temperatures in excess of 180.degree. C. Finally, the Guella et al. compounds include conjugated double bonds which exhibit a characteristic UV absorption (.lambda..sub.max) of 292. The lipid chemoattractant of this invention does not exhibit UV absorption at 292, indicating that the double bonds are not conjugated.